The present invention relates to a polypeptide possessing a ceramidase activity, an antibody specifically binding thereto, a gene encoding the polypeptide, and a probe and primer which are capable of specifically hybridizing thereto, which are useful as a reagent for lipid engineering for analyzing a structure, functions, and the like of a ceramide. In addition, the present invention relates to a method for producing the above-mentioned polypeptide by a genetic engineering means, and a method for detecting the polypeptide or the gene, and a kit therefor. Further, the present invention relates to a method of controlling an amount of a ceramide in a cell and/or in a tissue, which can be applied to a disease caused by abnormality in the amount of the ceramide.
A ceramidase is an enzyme which hydrolyzes a ceramide, a kind of a sphingolipid, into a sphingoid and a fatty acid. The sphingoid which is generated by hydrolyzing the ceramide with the ceramidase possesses various physiological activities such as inhibition of protein kinase C, activation of phospholipase D and inhibition of a calmodulin-dependent enzyme. As described above, the above-mentioned sphingoid is an important substance which is thought to be acting on the regulation of the cell functions because the sphingoid is involved in proliferation of cells and intracellular signal transduction. The ceramidase is an enzyme which plays an important role of the control of the amount of the above-mentioned sphingoid.
Ceramidases are classified into acidic ceramidases and neutral/alkaline ceramidases by the optimum pH. There have been, so far reported that the presence of a ceramidase possessing the optimum pH in an acidic range has been found in mammalian tissues such as rat brain [Biochemistry, 8, 1692-1698 (1969)], guinea pig epithelial cells [J. Biol. Chem., 270, 12677-12684 (1995)], human kidney [Biochim. Biophys. Acta, 398, 125-131 (1975)], spleen [Biochim. Biophys. Acta, 1004, 245-251 (1989)], fibroblasts [Biochem. J., 205, 419-425 (1982)], and epithelium [FEBS Lett., 268, 110-112 (1990)]; and human urine [J. Biol. Chem., 270, 11098-11102 (1995)], and the like.
In addition, it has been clarified that a bacterium belonging to the genus Pseudomonas produces a ceramidase, and this ceramidase is a ceramidase possessing optimum pH in an alkaline region [J. Biol Chem., 273, 14368-14373 (1998)].
Among these ceramidases, amino acid sequences of the acidic ceramidase purified from human urine and nucleotide sequences of a gene encoding the ceramidase have been determined [J. Biol Chem, 271, 33110-33115 (1996)]. In addition, an acidic ceramidase gene of a mouse has been obtained by utilizing its homology with the above-mentioned acidic ceramidase gene derived from human urine [Genomics, 50, 267-274 (1998)].
However, since all of the ceramidase genes derived from mammals which have been reported encode acidic ceramidases, amino acid sequences and genomic structures of the neutral/alkaline ceramidase in mammals have been completely unknown, so that biological functions of the neutral/alkaline ceramidase in higher organisms have not yet been elucidated at present.
In the studies on the elucidation of the in vivo functions of a ceramide, metabolic control therefor, diagnosis or treatment of a disease associated with the ceramide or the like, it is necessary to obtain detailed information concerning various enzymes associated with the ceramide, and the enzyme gene. However, as mentioned above, findings on the amino acid sequence and genes thereof of the neutral/alkaline ceramidase in mammals have not yet been obtained at present. Therefore, in order to develop the technique as described above pertaining to a ceramide, it is necessary to obtain some findings associated with a neutral/alkaline ceramidase, especially a gene thereof.
As mentioned above, although several reports have been made on cloning of ceramidase genes of a mammal, all of these reports are concerned with ceramidases possessing an activity in an acidic region, which cannot be expected to possess a homology with a ceramidase possessing an activity in a neutral/alkaline region. Therefore, it has been difficult to obtain a neutral/alkaline ceramidase gene as a homolog of a nucleotide sequence of the acidic ceramidase gene.
The present invention has been accomplished in view of the above-described prior art and a first object of the present invention is to provide a neutral/alkaline ceramidase gene of a mammal. A second object of the present invention is to provide a method for producing a neutral/alkaline ceramidase gene in a high purity by a genetic engineering means, comprising using a transformant resulting from incorporation of an expression vector carrying the gene. A third object of the present invention is to provide a polypeptide encoding the above-mentioned gene. A fourth object of the present invention is to provide an antisense DNA and an antisense RNA which are complementary to the gene of the present invention or a part thereof. A fifth object of the present invention is to provide a synthetic oligonucleotide probe or primer capable of specifically hybridizing to the gene of the present invention. A sixth object of the present invention is to provide an antibody or a fragment thereof specifically binding to the polypeptide. A seventh object of the present invention is to provide a method for detecting the above-mentioned ceramidase or a gene thereof, and a kit used therefor. An eighth object of the present invention is to provide a method of controlling an amount of ceramide in the cell or in the tissue.
The present inventors have succeeded in isolating a neutral/alkaline ceramidase from liver of a mouse, a mammalian, and isolating the genes. In addition, they have succeeded in elucidating the structure of the neutral/alkaline ceramidase of a mammal including human by using techniques such as hybridization or polymerase chain reaction (PCR). Further, they have also succeeded in conveniently producing a neutral/alkaline ceramidase in a high purity by genetic engineering techniques by using the genes. Thus, the present invention has been perfected thereby.
Specifically, the gist of the present invention relates to:
[1] a gene having a nucleotide sequence of a nucleic acid selected from the group consisting of:
(A) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 14 of the Sequence Listing or a partial sequence thereof, the polypeptide possessing a ceramidase activity;
(B) a nucleic acid having a nucleotide sequence of SEQ ID NO: 15 of the Sequence Listing or a partial sequence thereof and encoding a polypeptide possessing a ceramidase activity;
(C) a nucleic acid encoding a polypeptide consisting of an amino acid sequence resulting from deletion, addition, insertion or substitution of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 14 of the Sequence Listing, the polypeptide possessing a ceramidase activity;
(D) a nucleic acid consisting of a nucleotide sequence resulting from deletion, addition, insertion or substitution of at least one base in the nucleotide sequence of SEQ ID NO: 15 of the Sequence Listing and encoding a polypeptide possessing a ceramidase activity;
(E) a nucleic acid capable of hybridizing to a complementary strand of a nucleic acid of any one of the above (A) to (D), under stringent conditions, and encoding a polypeptide possessing a ceramidase activity; and
(F) a nucleic acid having a nucleotide sequence different from the nucleic acid of any one of above (A) to (E) via degeneracy and encoding a polypeptide possessing a ceramidase activity;
[2] a recombinant DNA comprising the gene of item [1] above;
[3] an expression vector for a microorganism, an animal cell or a plant cell, comprising the gene of item [1] above or the recombinant DNA of item [2] above;
[4] a transformant carrying the expression vector of item [3] above;
[5] a method for producing a polypeptide possessing a ceramidase activity, characterized by culturing the transformant of item [4] above under conditions appropriate for expression of the ceramidase gene and production of the polypeptide encoded by the gene, and collecting a polypeptide possessing a ceramidase activity from the resulting culture;
[6] a polypeptide having the amino acid sequence of SEQ ID NO: 14 of the Sequence Listing or a partial sequence thereof and possessing a ceramidase activity;
[7] a polypeptide possessing a ceramidase activity, encoded by the gene of item [1] above;
[8] an antisense DNA which is complementary to the gene of item [1] above or a part thereof;
[9] an antisense RNA which is complementary to the gene of item [1] above or a part thereof;
[10] an expression vector comprising the antisense DNA of item [8] above;
[11] an oligonucleotide probe or primer, capable of specifically hybridizing to the gene of item [1] above or a complementary strand thereof;
[12] an antibody or a fragment thereof, capable of specifically binding to the polypeptide of item [6] or item [7] above;
[13] a method for detecting a gene encoding a polypeptide possessing a ceramidase activity, comprising using the oligonucleotide probe or primer of item [11] above;
[14] a kit for the use in detection of a gene encoding a polypeptide possessing a ceramidase activity, comprising the oligonucleotide probe and/or primer of item [11] above;
[15] a method for detecting a polypeptide possessing a ceramidase activity, comprising using the antibody or a fragment thereof of item [12] above;
[16] a kit for the use in detection of a polypeptide possessing a ceramidase activity, comprising the antibody or a fragment thereof of item [12] above; and
[17] a method of controlling an amount of ceramide in a cell and/or in a tissue, characterized by introducing the gene of item [1] above or an antisense nucleic acid thereof into the cell and/or the tissue, thereby controlling the amount of ceramide in the cell and/or in the tissue.